Physiology
Systemic activation of the coagulation system is frequently observed in patients with severe sepsis and/or septic shock. Severe sepsis, as a complication of infection, is characterized by systemic inflammation, activation of proteolytic cascades, coagulation abnormalities (DIC), and various organ dysfunctions. Its more severe form, septic shock, associates in addition altered hemodynamic and impaired organ perfusion, aggravating further organ failure and frequently leading to death in multiorgan failure. Mortality in septic shock is high (40% to 50%), and rises tremendously with the number of failing organs, i.e. patients requiring renal replacement therapy for acute renal failure may have mortality rate above 80%. The initiating event for the development of severe sepsis is the activation of monocytes/macrophages by microbial antigens (i.e. lipopolysaccharide (LPS) liberated from Gram-negative bacteria, lipoteichoic acid from Gram-positive bacteria, fungal antigen) via binding to surface Toll-like receptors (Cohen J. The immunopathogenesis of sepsis. Nature. 2002; 420:885-91).
Mechanistic
Monocyte/macrophages activation, potentially followed by endothelial cells activation, results in secretion of cytokine mediators (such as interleukin 6 (IL-6), tumor necrosis factor (TNFα) and chemokines, lipid mediators, adhesion molecules and tissue factor expression at the cell membrane, nitric oxyde and oxygen reactive species formation). This step is followed by the activation of biological cascades including the coagulation and the complement, which contribute to the maintenance of the inflammatory reaction and coagulation activation. These events are fully adapted to eradicate microbial agents at the site of infection, but results in deleterious consequences, i.e. various organ failures, if they become widespread, unregulated and sustained. (Aird W. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood. 2003; 101:3765-77).
Tissue factor expression leads to uncontrolled generation of thrombin from its precursor molecule prothrombin. Endogenous anticoagulants, namely antithrombin (AT), protein C pathway and Tissue Factor Pathway Inhibitor, as well as the fibrinolytic system are activated and function to regulate thrombin generation and its consequences. However, blood levels of endogenous inhibitor are diminished due to their consumption onto their substrate, inhibited synthesis and cleavage by several proteases. Indeed, AT levels decrease precipitously in the early phases of severe sepsis. Fibrinolysis is rapidly inhibited by the production of plasminogen activator inhibitor-1. Thus, a procoagulant state develops in patients with sepsis potentially leading to overt disseminated intravascular coagulation (DIC), which has deleterious effects. Diffuse thrombus formation throughout microcirculation can compromise tissue perfusion to critical organs, and cellular effects of various coagulation proteins participate in the general inflammatory syndrome (Aird W. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood. 2003; 101:3765-77). It is demonstrated that coagulation abnormalities in severe sepsis is associated with poor outcome. Notably, depletion of AT in septic shock portends a poor prognosis (Fourrier F. Septic shock, multiple organ failure, and disseminated intravascular coagulation: compared patterns of antithrombin III, protein C, and protein S deficiencies. Chest. 1992; 101:816-823).
Antithrombin
Antithrombin plays an essential role in maintaining the fluidity of blood. Blood coagulation is mediated by a series of serine proteases. Antithrombin is a potent inhibitor of coagulation serine proteases, in particular, Factors IIa (thrombin), Xa, IXa and XIa. Interaction of AT with heparin-like glycosaminoglycans (HGAGs) on endothelial cells is important for the acceleration of thrombin inhibition by AT (Rosenberg R D. Biochemistry of heparin antithrombin interactions, and the physiologic role of this natural anticoagulant mechanism. Am J Med. 1989; 87:2S-9S). Indeed, thrombotic episodes have been observed in patients with congenital AT deficiency and in those with variant AT that lacks affinity for heparin suggesting that the interaction of AT with the endothelial cell surface heparin-like GAGs is important for regulation of the coagulation cascade by AT (Kuhle S, Lane D A, Jochmanns K, Male C, Quehenberger P, Lechner K, Pabinger I. Homozygous antithrombin deficiency type II (99 Leu to Phe mutation) and childhood thromboembolism. Thromb Haemost. 2001; 86:1007-11). The physiological importance of antithrombin in preventing excessive coagulation is revealed by studies of individuals whose antithrombin levels are decreased due to heredity or acquired deficiency. Such persons are prone spontaneous thrombosis and the associated risks of disseminated intravascular coagulation, cardiac infarction, cerebrovascular accident and pulmonary embolism.
In addition, AT has been shown to exert cytoprotective properties, through its binding to the glycosaminoglycans present on endothelial cells surface Indeed, AT inhibits NFkB activation endothelial cells and monocytes, resulting in an increase in prostacyclin synthesis, a decrease in pro-inflammatory cytokines production (IL6 and TNFα), a reduction in tissue factor exposure on endothelial cells or monocytes, a reduction in platelet aggregation and in endothelial cells/neutrophil interactions. This effect is abolished by heparin, through a competition process. These cytoprotective effects were observed for AT concentrations ranging from 2.5 to 40 UI/ml (corresponding to 2.5 to 40 fold normal circulating AT levels).
Models of Sepsis
AT has been shown to be efficacious in several experimental models of sepsis and septic shock, regardless of the species investigated, as shown in baboons, dogs, sheep, rabbits, rats, and chicken embryos. AT in these models proved to be effective after inducing a sepsis or septic shock with different agents including live bacteria (Escherichia coli, Klebsiella pneumoniae) and bacterial lipopolysaccharide (LPS).
In a guinea pig model, it has been demonstrated that AT could prevent DIC and organ hemorrhage and improve mortality after infection with the Gram-positive bacterium Staphylococcus aureus. 
Due to the missregulation of coagulation process during sepsis, some anticoagulants have been assayed in human, such as heparin or antithrombin.
Therapeutic doses of heparin (Corrigan J J. Heparin therapy in bacterial septicemia. J Pediatr. 1977; 91:695-700) or antithrombin (Schipper H G, Jenkins C S P, Kahl L H, ten Cate J W. Antithrombin III transfusion in disseminated intravascular coagulation. Lancet. 1978; 1:854-856) have been used clinically for more than 20 years for the prevention and treatment of disseminated intravascular coagulation and sepsis, without any clear evidence of efficacy.
In order to treat activation of coagulation system and depletion of anticoagulant during severe sepsis, some studies have been made in patients by using high doses of antithrombin.
Warren et al. (High dose Antithrombin III in severe sepsis, 2008, JAMA, 286(15), 1869-1878) have tested the effect of administration of 30,000 IU (cumulative dose for a 4-day treatment) AT in patients in a randomized controlled trial study. Patients receiving AT treatment had plasma AT levels around 180% of normal circulating blood levels, these levels being lower than doses required to achieve a cytoprotective effect. This study could not demonstrate the efficiency of AT administration on patient survival. Moreover, the authors have demonstrated that the administration of AT, at this dosage, enhances the hemorrhagic risk in patients, said risk being increased when patient has received a concomitant administration of heparin.
A post-hoc analysis of this study focusing on DIC related to severe sepsis reported that this dosage of AT (30,000 IU over 4 days) increases the hemorrhagic risk in patients without heparinic treatment, compared to control treated with a placebo (Treatment effects of high-dose antithrombin without concomitant heparin in patients with severe sepsis with or without disseminated intravascular coagulation, 2006, J. of Thrombosis and Haemostasis, 4: 90-97),
Another study (Eisele at al. Antithrombin III in patients with severe sepsis, intensive care Med., 1998, 24:663-672) has demonstrated that 18,000 UI (cumulative dose for a 5-day treatment) of AT have some benefits in patient survival, without bleeding problems. However, the panel of patients used in this study is too small to be sure that this dosage of AT has no effect on bleeding, and the dosage of AT remains under the identified efficient dosage of antithrombin necessary to provide a good cytoprotective effect: indeed, in this study, patients receiving AT treatment had AT levels ranging from 70 to 130% of normal circulating AT levels.
So there is a need to provide new medicines for treating severe sepsis, which enhances the anticoagulation system in the patient, but without causing hemorrhagic accidents.
Other anticoagulant has been tested in the treatment of severe sepsis.
Activated protein C (drotrecogin α) has been shown to reduce severe sepsis mortality in patients with the highest severity scores [Bernard G R, Vincent J L, Laterre P F, LaRosa S P, Dhainaut J F, Lopez-Rodriguez A, Steingrub J S, Garber G E, Helterbrand J D, Ely E W, Fisher C J Jr; Recombinant human protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344:699-709]. In spite of the results of this study, the use of activated protein C remains controversial, as controversy regarding the efficacy/safety profile of this drug notably regarding bleeding. WO2005/007820 discloses the use of a mutated activated protein C variants, with reduced anticoagulant activity, for the treatment of pathologies requiring a cellular cytoprotection, but these variants have not been tested in clinical practice [Kerschen E J, Fernandez J A, Cooley B C, Yang X V, Sood R, Mosnier L O, Castellino F J, Mackman N, Griffin J H, Weiler H. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exp Med. 2007 Oct. 1; 204(10):2439-48.].
Thus, it is important to provide a new medicine that can cure all the forms of severe sepsis, without causing any, or reduced only, hemorrhagic manifestation in patients.